PROJECT SUMMARY This project proposes a comprehensive five-year mentored research development program with transition to independence. The research plan investigates the severe clinical problem of intestinal fibrosis and stricture formation in inflammatory bowel diseases (IBD). It explores the entirely novel concept of a direct effect of bacterial components on intestinal mesenchymal cells, the chief pro-fibrotic cell type, in vitro and in vivo. We found that human intestinal myofibroblasts respond to activation of bacterial sensing pattern recognition receptors, but only flagellin increases secretion of the extracellular matrix (ECM) components fibronectin and collagen 1. This response is post-transcriptionally regulated and dependent on caspase 1, implicating the inflammasome in intestinal fibrosis. Therefore, we propose to explore the following hypothesis: the gut microbiota induces intestinal fibrosis through a flagellin mediated pathway. This hypothesis will be tested through three interrelated, but indepedent specific aims: (1) to define the mechanisms of flagellin- induced post-transcriptional regulation of ECM secretion in vitro (2) to determine the role of flagellin mediated inflammasome activation in the pro-fibrogenic responses in primary human mesenchymal cells in vitro and (3) to explore the effect of abrogating flagellin signaling in experimental fibrosis in vivo during induction and resolution of intestinal fibrosis. Post-transcriptional regulation and inflammasome activation via caspase 1 are two entirely original mechanisms of intestinal fibrosis. We will use a cutting edge transgenic mouse model that allows control of the timing of deletion of bacterial sensing in vivo specifically in mesenchymal cells. This enables exploration of microbial sensing in the prevention and resolution of intestinal fibrosis. The research will be carried out in the laboratory of Dr. Fiocchi MD, Lerner Research Institute (LRI), Cleveland Clinic, and will be advised by a panel of international experts in human and experimental fibrogenesis, translational regulation and the microbiota. The advisory panel has worked out a structured career development program, including formal coursework in microbiology, translational regulation and statistics. An ideal intellectual and technical environment is in place locally in the LRI. My career goal is to build and lead an independent research program that will advance scientific knowledge and patient care in the field of intestinal fibrosis. My prior experience with primary human cell systems and animal models gives me the ideal basis to be a successful K08 awardee. The proposed study will provide novel evidence for the direct effect of the microbiota in intestinal mesenchymal cell induced fibrogenesis and stricture formation. These findings could lead to a completely novel approach of targeting the microbiota as a therapeutic strategy to abrogate fibrosis.